Detecting single nucleotide polymorphism using hydrolysis probes with 3′ hairpin structure

ABSTRACT

SNP specific hydrolysis probe including a hairpin structure toward the 3′ end, along with kits are provided that are designed for the detection of a SNP in a target nucleic acid.

CROSS REFERENCE TO RELATED INVENTION

This application is a divisional of U.S. application Ser. No. 14/106,456filed on Dec. 13, 2013, now U.S. Pat. No. 9,297,033, the content ofwhich is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “31771_US1_Sequence_Listing.txt”, having a size in bytesof 3 kb, and created on Feb. 18, 2016. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to the field of polymerase chain reaction(PCR) based diagnostic, and more particularly, to PCR detection methodsutilizing hydrolysis probes.

BACKGROUND OF THE INVENTION

PCR is an efficient and cost effective way to copy or ‘amplify’ smallsegments of DNA or RNA. Using PCR, millions of copies of a section ofDNA are made in just a few hours, yielding enough DNA required foranalysis. This method allows clinicians to diagnose and monitor diseasesusing a minimal amount of sample, such as blood or tissue. Real-time PCRallows for amplification and detection to occur at the same time. Onemethod of detection is done by utilizing oligonucleotide hydrolysisprobes (also known as TaqMan® probes) having a fluorophore covalentlyattached, e.g., to the 5′ end of the oligonucleotide probe and aquencher attached, e.g., internally or at the 3′ end. Hydrolysis probesare dual-labeled oligonucleotide probes that rely on the 5′ to 3′exonuclease activity of Taq polymerase to cleave the hydrolysis probeduring hybridization to the complementary target sequence, and result influorescent based detection.

Real time PCR methods can be used for amplifying and detecting sequencevariations in target nucleic acids having single nucleotide polymorphism(SNP). However, many of the available SNP detection/genotyping assaysare based on the assumption that the SNP is biallelic (see, e.g., Moritaet al., Mol. Cel. Probes, 2007, 21, 171-176). Detection of SNP withcurrently existing real time PCR methods lacks sufficient sensitivityand specificity. Hydrolysis probes, such as standard TaqMan® probes, aretypically designed to be about 18 to 22 bases in length in order to have8-10° C. higher melting temperature (Tm) as compared to the primer.Standard TaqMan® probes generally prove to be less specific andsensitive for SNP detection and fail to show complete discriminationbetween the WT (Wild-type) and the MT (Mutant) targets. Current TaqMan®based SNP genotyping assays involve the use of TaqMan® MGB (Minor GrooveBinders) probes that are shorter in length with increased probe-templatebinding stability for allelic discrimination. Additional basemodifications such as stabilizing bases (propynyl dU, propynyl dC) canalso be included in standard TaqMan® probe design for improved SNPdetection and discrimination. Thus there is a need in the art for aquick and reliable method to specifically detect SNPs in a sensitivemanner.

SUMMARY OF THE INVENTION

The subject matter of the present disclosure includes SNP specifichydrolysis probes which are designed to include a hairpin structuretoward the 3′ end. Such hydrolysis probes do not involve the use ofadditional base modifications such as propynyl dU, propynyl dC, orspecial molecules such as MGBs. The hairpin structure near the 3′ end ofthe probe delays the hybridization of the 3′ portion of the probe to thetemplate and thus helps in the discrimination of the WT and the MTtargets based on the single mismatch between the reporter and thequencher which is near the 5′ end. The 5′ portion of the SNP specificprobe can hybridize more efficiently to the MT template as compared tothe WT template. When the SNP specific probe finds the WT target, thesingle mismatch to the WT target can prevent hybridization and probecleavage, and thus no fluorescence can be detected.

In one embodiment, a method for detecting a SNP in a target nucleic acidin a sample is provided, the method including performing an amplifyingstep comprising contacting the sample with a primer comprising a firstnucleic acid sequence to produce an amplification product if any targetnucleic acid is present in the sample; performing a hybridizing stepcomprising contacting the amplification product with a SNP specifichydrolysis probe comprising a second nucleic acid sequence complementaryto a SNP containing region of the amplification product, the SNPspecific hydrolysis probe comprising a first and a second interactivelabel, a 5′ end and a 3′ end, and a hairpin structure toward the 3′ end,the hairpin structure comprising a region of non-naturally occurringnucleic acid sequence comprising one or more non-naturally occurring(e.g., changed or additional) nucleotides to produce the hairpinstructure; and detecting the presence or absence of the amplificationproduct, wherein the presence of the amplification products isindicative of the presence of the SNP in the target nucleic acid target,and wherein the absence of the amplification products is indicative ofthe absence of the SNP in the target nucleic acid target.

In another embodiment, a kit for detecting a SNP in a target nucleicacid in a sample is provided, including at least one primer including afirst nucleic acid sequence specific to produce an amplification productof the target nucleic acid; and a SNP specific hydrolysis probecomprising a second nucleic acid sequence complementary to a SNPcontaining region of the amplification product, the SNP specifichydrolysis probe comprising a first and a second interactive label, a 5′end and a 3′ end, and a hairpin structure toward the 3′ end, the hairpinstructure comprising a region of non-naturally occurring nucleic acidsequence comprising one or more non-naturally occurring (e.g., changedor additional) nucleotides to produce the hairpin structure. The kit mayalso include a polymerase enzyme having 5′ to 3′ exonuclease activity.

In one embodiment, a SNP specific hydrolysis probe is provided includinga nucleic acid sequence complementary to a SNP containing region of theamplification product, the SNP specific hydrolysis probe comprising afirst and a second interactive label, a 5′ end and a 3′ end, and ahairpin structure toward the 3′ end, the hairpin structure comprising aregion of non-naturally occurring nucleic acid sequence comprising oneor more non-naturally occurring (e.g., changed or additional)nucleotides to produce the hairpin structure. The first interactivelabel may be a donor fluorescent moiety toward, near, or at the 5′terminus, and the second interactive label may be a correspondingacceptor fluorescent moiety, e.g., a quencher, for example, within nomore than 5 nucleotides of the donor fluorescent moiety on thehydrolysis probe.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows real time PCR amplification curves for 526N SNP detectionusing hydrolysis probe with a hairpin structure toward the 3′ end.

FIG. 2 shows location of 526N SNP (CAC/AAC) on a portion of a MT plasmid(SEQ ID NO: 5) and WT plasmid (SEQ ID NO: 6) and a probe without hairpin(long arrow) with a terminal base replaced to prevent intermolecularinteractions.

FIG. 3 shows sequence (SEQ ID NO: 1) of a hydrolysis probe for 526N SNPlocation designed with a hairpin structure at the 3′ end having threebases replaced to form the hairpin.

FIG. 4 shows real time PCR amplification curves for 526N SNP detectionusing hydrolysis probe without a hairpin structure.

FIG. 5 shows real time PCR amplification curves for 531L SNP detectionusing hydrolysis probe with a hairpin structure toward the 3′ end.

FIG. 6 shows location of 531L SNP (TCG/TTG) on a portion of a MT plasmid(SEQ ID NO: 7) and WT plasmid (SEQ ID NO: 8) and a probe without hairpin(long arrow).

FIG. 7 shows sequence (SEQ ID NO: 3) of a hydrolysis probe for 531L SNPlocation designed with a hairpin structure at the 3′ end having one basereplaced to form the hairpin.

FIG. 8 shows real time PCR amplification curves for 531L SNP detectionusing hydrolysis probe without a hairpin structure.

DETAILED DESCRIPTION OF THE INVENTION

Methods, kits, and hydrolysis probes for detecting a single nucleotidepolymorphism (SNP) in a target nucleic acid in a sample are describedherein. The increased sensitivity of real-time PCR for detection of aSNP in a target nucleic acid compared to other methods, as well as theimproved features of real-time PCR including sample containment andreal-time detection of the amplified product, make feasible theimplementation of this technology for routine diagnosis and detection ofa SNP in a target nucleic acid in the clinical laboratory.

The methods may include performing at least one cycling step thatincludes amplifying one or more portions of a target nucleic acidmolecule, e.g., a gene target containing the SNP of interest to bedetected, in a sample using one or more primers or one or more primerpairs. As used herein, “primer”, “primers”, and “primer pairs” refer tooligonucleotide primer(s) that specifically anneal to the nucleic acidsequence target, and initiate synthesis therefrom under appropriateconditions. Each of the primers anneal to a region within or adjacent tothe respective target nucleic acid molecule such that at least a portionof each amplification product contains nucleic acid sequencecorresponding to respective target and SNP, if present. An amplificationproduct is produced provided that the target nucleic acid is present inthe sample, whether or not the SNP of interest is present in the targetnucleic acid molecule.

The method can also include a hybridizing step that includes contactingthe amplification product with a SNP specific hydrolysis probe includinga nucleic acid sequence complementary to a SNP containing region of theamplification product. The SNP specific hydrolysis probe can include afirst and a second interactive label, a 5′ end and a 3′ end, and ahairpin structure toward the 3′ end. The hairpin structure can bedesigned to include a nucleic acid region that is non-naturallyoccurring which may include one or more changed nucleotides that are notpart of the naturally occurring sequence, or may include one or moreadditional non-naturally occurring nucleotides, which are nucleotidesadded to the naturally occurring sequence, in order to produce thehairpin structure. In this way, a nucleic acid sequence that does notnormally form a hairpin structure at the 3′ end can be designed to forma hairpin by, e.g., altering the nucleic acid sequence, for example,changing one or more nucleotides in the sequence toward the 3′ end, orby adding one or more nucleotides to the nucleic acid sequence at the 3′end.

In order to detect whether or not the SNP of interest is present orabsent in the nucleic acid target in the sample, the amplificationproduct is detected by way of the detectable label being released fromthe SNP specific hydrolysis probe. If the amplification product isdetected by way of the SNP specific hydrolysis probe, the presence ofSNP is indicated. If alternatively, the amplification product is notdetected by way of the SNP specific hydrolysis probe, the presence ofSNP is not indicated. Thus, the presence of the amplification productsis indicative of the presence of the SNP in the target nucleic acidtarget, and the absence of the amplification products is indicative ofthe absence of the SNP in the target nucleic acid target.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., target nucleicacid molecules for Human immunodeficiency virus (HIV) or Mycobacteriumtuberculosis (MTB), or Hepatitis C virus (HCV)). Amplifying a nucleicacid molecule typically includes denaturing the template nucleic acid,annealing primers to the template nucleic acid at a temperature that isbelow the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product.Amplification typically requires the presence of deoxyribonucleosidetriphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and anappropriate buffer and/or co-factors for optimal activity of thepolymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “primer” is used herein as known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group whereto further“nucleotides” may be attached by a template-dependent DNA polymeraseestablishing 3′ to 5′ phosphodiester linkage whereby deoxynucleosidetriphosphates are used and whereby pyrophosphate is released. Ingeneral, primers are designed based on known template sequences. Oneprimer primes the sense strand, and the other primes the complementarystrand of the target DNA or cDNA. PCR can be performed on a uniformtarget DNA or RNA (i.e., targets with the same sequence) or on mixedtarget DNAs or RNAs, (i.e., targets with different intervening sequencesflanked by conserved sequences). For mixed DNAs/RNAs (e.g., containingsequence heterogeneity) even mismatched primers can function in the PCRreaction if the sequences of the targets have enough complementarity tothe mismatched primers (i.e., tolerant primers).

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ exonuclease activity” refers to an activity of anucleic acid polymerase, typically associated with the nucleic acidstrand synthesis, whereby nucleotides are removed from the 5′ end ofnucleic acid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus fiavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., aC5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides of theinvention are referred to herein or are otherwise known in the art. Incertain embodiments, modified nucleotide substitutions modify meltingtemperatures (Tm) of the oligonucleotides relative to the meltingtemperatures of corresponding unmodified oligonucleotides. To furtherillustrate, certain modified nucleotide substitutions can reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like in some embodiments of the invention. Examplesof these types of nucleic acid modifications are described in, e.g.,U.S. Pat. No. 6,001,611, which is incorporated herein by reference.

Target Nucleic Acids and Oligonucleotides

The present description provides methods to detect SNP in a targetnucleic acid by amplifying, for example, a portion of the target nucleicacid sequences, which may be any target nucleic acid sequence the inknown or suspected to comprise one or more SNPs, for example targetnucleic acid sequences from, e.g., HIV, HCV, or MTB that is rifampicinresistant.

For detection of SNP in the target nucleic acid sequence, primers andprobes to amplify the target nucleic acid sequences can be prepared.Also, functional variants can be evaluated for specificity and/orsensitivity by those of skill in the art using routine methods.Representative functional variants can include, e.g., one or moredeletions, insertions, and/or substitutions in the primers and/or probesdisclosed herein. For example, a substantially identical variant of theprimers or probes can be provided in which the variant has at least,e.g., 80%, 90%, or 95% sequence identity to one original primers andprobes, or a complement thereof.

A functionally active variant of any of primer and/or probe may beidentified which provides a similar or higher specificity andsensitivity in the presently described methods, kits, or hydrolysisprobes as compared to the respective original sequences.

As detailed above, a primer (and/or probe) may be chemically modified,i.e., a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify the target nucleic acid sequences can be designedusing, for example, a computer program such as OLIGO (Molecular BiologyInsights Inc., Cascade, Colo.). Important features when designingoligonucleotides to be used as amplification primers include, but arenot limited to, an appropriate size amplification product to facilitatedetection (e.g., by electrophoresis), similar melting temperatures forthe members of a pair of primers, and the length of each primer (i.e.,the primers need to be long enough to anneal with sequence-specificityand to initiate synthesis but not so long that fidelity is reducedduring oligonucleotide synthesis). Typically, oligonucleotide primersare 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

In addition to a set of primers, the present methods may use one or moreprobes in order to detect the presence or absence of SNP in a targetnucleic acid sequence. The term “probe” refers to synthetically orbiologically produced nucleic acids (DNA or RNA), which by design orselection, contain specific nucleotide sequences that allow them tohybridize under defined predetermined stringencies specifically (i.e.,preferentially) to “target nucleic acids”. A “probe” can be referred toas a “detection probe” meaning that it detects the target nucleic acid.

In some embodiments of the present invention, the described probes canbe labeled with at least one fluorescent label. In one embodiment probescan be labeled with a donor fluorescent moiety, e.g., a fluorescent dye,and a corresponding acceptor fluorescent moiety, e.g., a quencher.

Designing oligonucleotides to be used as TaqMan hydrolysis probes can beperformed in a manner similar to the design of primers. Embodiments ofthe present invention may use a single probe for detection of theamplification product. Depending on the embodiment, the probe mayinclude at least one label and/or at least one quencher moiety. As withthe primers, the probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18,20, 21, 22, 23, 24, or 25) nucleotides in length.

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188 discloseconventional PCR techniques. U.S. Pat. Nos. 5,210,015; 5,487,972;5,804,375; 5,804,375; 6,214,979; and 7,141,377 disclose real-time PCRand TaqMan® techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the described embodiments includeoligonucleotides capable of acting as a point of initiation of nucleicacid synthesis within the target nucleic acid sequences. A primer can bepurified from a restriction digest by conventional methods, or it can beproduced synthetically. The primer is preferably single-stranded formaximum efficiency in amplification, but the primer can bedouble-stranded. Double-stranded primers are first denatured, i.e.,treated to separate the strands. One method of denaturing doublestranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the target nucleicacid molecules. The temperature for annealing is usually from about 35°C. to about 65° C. (e.g., about 40° C. to about 60° C.; about 45° C. toabout 50° C.). Annealing times can be from about 10 sec to about 1 min(e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). Thereaction mixture is then adjusted to a temperature at which the activityof the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the template nucleic acid. The temperatureshould be sufficient to synthesize an extension product from each primerthat is annealed to a nucleic acid template, but should not be so highas to denature an extension product from its complementary template(e.g., the temperature for extension generally ranges from about 40° C.to about 80° C. (e.g., about 50° C. to about 70° C.; about 60° C.).Extension times can be from about 10 sec to about 5 min (e.g., about 30sec to about 4 min; about 1 min to about 3 min; about 1 min 30 sec toabout 2 min).

PCR assays can employ target nucleic acid such as RNA or DNA (cDNA). Thetemplate nucleic acid need not be purified; it may be a minor fractionof a complex mixture, such as target nucleic acid contained in humancells. Target nucleic acid molecules may be extracted from a biologicalsample by routine techniques such as those described in DiagnosticMolecular Microbiology: Principles and Applications (Persing et al.(eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acid molecules. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength.

In one example, a oligonucleotide probe can contain a donor fluorescentmoiety and a corresponding quencher, which dissipates the transferredenergy in a form other than light. When the probe is intact, energytransfer typically occurs between the two fluorescent moieties such thatfluorescent emission from the donor fluorescent moiety is quenched.During an extension step of a polymerase chain reaction, a probe boundto an amplification product is cleaved by the 5′ to 3′ exonucleaseactivity of, e.g., a Taq polymerase such that the fluorescent emissionof the donor fluorescent moiety is no longer quenched. Exemplary probesfor this purpose are described in, e.g., U.S. Pat. Nos. 5,210,015;5,994,056; and 6,171,785. Commonly used donor-acceptor pairs include theFAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonlyused dark quenchers include BlackHole Quenchers™ (BHQ), (BiosearchTechnologies, Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech.,Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650), (Berry &Assoc., Dexter, Mich.).

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present disclosure is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 Å to about 25 Å. The linker armmay be of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640-NHS-ester, can becombined with C6-Phosphoramidites (available from ABI (Foster City,Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LCRed 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of a SNP in a Target Nucleic Acid

The present disclosure provides methods for detecting the presence orabsence of a SNP in a target nucleic acid in a biological. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of the target nucleic acidmolecule from a sample using a primer pair, and a fluorescent detectingstep utilizing hydrolysis probes having a hairpin structure toward the3′ end. Multiple cycling steps may be performed, preferably in athermocycler. The described methods can be performed using the primersand probes to detect the presence of the SNP in a target nucleic acid inthe sample.

As described herein, amplification products can be detected usinglabeled hydrolysis probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof a SNP in a target nucleic acid. TaqMan® technology utilizes onesingle-stranded hybridization hydrolysis probe labeled with twofluorescent moieties. When a first fluorescent moiety is excited withlight of a suitable wavelength, the absorbed energy is transferred to asecond fluorescent moiety according to the principles of FRET. Thesecond fluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems) uses TaqMan® technology, and is suitable for performing themethods described herein for detecting the presence or absence of a SNPin a target nucleic acid in the sample.

Generally, the presence of FRET indicates the presence of the SNP in atarget nucleic acid in the sample, and the absence of FRET indicates theabsence of HSV-1 and/or HSV-2 in the sample. Inadequate specimencollection, transportation delays, inappropriate transportationconditions, or use of certain collection swabs (calcium alginate oraluminum shaft) are all conditions that can affect the success and/oraccuracy of a test result, however. Using the methods disclosed herein,detection of FRET within, e.g., 45 cycling steps is indicative of thepresence of an SNP in a target nucleic acid in a sample.

Representative biological samples that can be used in practicing themethods of the invention include, but are not limited to dermal swabs,nasal swabs, wound swabs, blood cultures, skin, and soft tissueinfections. Collection and storage methods of biological samples areknown to those of skill in the art. Biological samples can be processed(e.g., by nucleic acid extraction methods and/or kits known in the art)to release target nucleic acid or in some cases, the biological samplecan be contacted directly with the PCR reaction components and theappropriate oligonucleotides.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples. Each thermocycler run canalso include a negative control that, for example, lacks target templateDNA. Such controls are indicators of the success or failure of theamplification, hybridization, and/or FRET reaction. Therefore, controlreactions can readily determine, for example, the ability of primers toanneal with sequence-specificity and to initiate elongation, as well asthe ability of probes to hybridize with sequence-specificity and forFRET to occur.

In an embodiment, the methods of the invention include steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996; 5,683,896; and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods of the invention. In one embodiment, aLightCycler® instrument is used. The following patent applicationsdescribe real-time PCR as used in the LightCycler® technology: WO97/46707, WO 97/46714, and WO 97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

It is understood that the embodiments of the present invention are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

The present disclosure further provides for articles of manufacture orkits to detect a SNP in a target nucleic acid. An article of manufacturecan include primers and probes used to detect the SNP, together withsuitable packaging materials. Representative primers and probes fordetection of the SNP are capable of hybridizing to the target nucleicacid molecules. In addition, the kits may also include suitably packagedreagents and materials needed for DNA immobilization, hybridization, anddetection, such solid supports, buffers, enzymes, and DNA standards.Methods of designing primers and probes are disclosed herein, andrepresentative examples of primers and probes that amplify and hybridizeto a SNP in a target nucleic acid target nucleic acid molecules areprovided.

Articles of manufacture of the invention can also include one or morefluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor and/or an acceptor fluorescent moietyfor labeling the SNP specific probes. Examples of suitable FRET donorfluorescent moieties and corresponding acceptor fluorescent moieties areprovided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the primers and probes todetect a SNP in a target nucleic acid in a sample. Articles ofmanufacture may additionally include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes, co-factors,or agents to prevent contamination). Such reagents may be specific forone of the commercially available instruments described herein.

Embodiments of the present invention will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example I MTB-RIF TaqMan® SNP Detection

Tuberculosis (TB) is a serious lung disorder commonly caused byMycobacterium tuberculosis (MTB) or other members of the MTB-complex.Drug-resistant strains of MTB are on the rise and a particularlydangerous form of drug-resistant tuberculosis is multidrug-resistanttuberculosis (MDR-TB). MDR-TB is defined as MTB that has developedresistance to at least two of the most commonly used anti-tuberculosisdrugs, rifampicin and isoniazid. Approximately 83-87% of the rifampicinresistance is caused by single nucleotide polymorphism within the 81base pair Rifampicin Resistance Determining Region (RRDR) of the rpoBgene encoding the β-subunit of RNA polymerase.

Mutant specific TaqMan® hydrolysis probes were designed with afluorophore at the 5′ end and an internal quencher. In addition to that,additional base/bases were introduced at the 3′ end of the probe thatwould result in a hairpin structure towards the 3′ end. Probe wasdesigned so that the mismatch between the WT and MT lies between thereporter and the quencher near the 5′ end. When the TaqMan® probe isintact, the reporter and quencher stay close to each other, whichprevent the emission of any fluorescence.

The primer and TaqMan® probe anneal to the complementary DNA strandfollowing denaturation during PCR. After hybridization and during theextension phase of PCR, the 5′ to 3′ exonuclease activity of the DNApolymerase cleaves the probe which separates reporter and quencher dyesand fluorescence is detected. Hairpin structure near the 3′ end of theprobe delays the hybridization of the 3′ half of the probe to thetemplate and thus helps in the discrimination of the WT and the MTtarget based on the single base pair difference or mismatch. The 5′ halfof the MT hairpin TaqMan® probe will hybridize more efficiently to theMT plasmid DNA template as compared to the WT template. When the MTspecific probe finds the WT target, the single mismatch to the WT targetwill prevent hybridization and probe cleavage and no fluorescence isdetected.

Materials and Methods

DNA Target-Wild-TYPE (WT) and Mutant (MT) Plasmids

MT and WT plasmid DNA: tested inputs ranging from 1e6 cp/PCR to 10cp/PCR shown in Table I showing a portion of the Rifampicin ResistanceDetermining Region (RRDR) of the rpoB gene in Mycobacteriumtuberculosis.

TABLE I Wildtype and Mutant Plasmid DNA SEQ ID NO SEQUENCE 5 MT PlasmidGCCAGCTGAGCCAATTCATGGTCCAGAAC with 526N AACCCGCTGTCGGGGTTGACCAACAAGCGSNP CCGACTGTCGGCGCTGGGGTCCGGCGG 6 WT PlasmidGCCAGCTGAGCCAATTCATGGACCAGAAC AACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGG 7 MT Plasmid GCCAGCTGAGCCTATTCATGGACCAGAACfor AACCCGCTGCAGGGGTTGACCCACAAGCG 531L SNP CCGACTGTTGGCGCTGGGGCCCGGCGG 8WT Plasmid GCCAGCTGAGCCAATTCATGGACCAGAAC AACCCGCTGTCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTGGGGCCCGGCGG

MTB Specific Oligonucleotides: One Set of Forward and Reverse Primersfor Both Wild-Type and Mutant Plasmids

Mutant specific hairpin TaqMan® probes shown in Table II

TABLE II SNP Specific Probes with and without hairpin structure SEQID NO SEQUENCE 1 Probe (526N 5′-FTTGTTGQGTCAACCCCG SNP) with 3′ AC GG GG P-3′ hairpin 2 Probe (526N 5′-FTTGTTGQGTCAACCCCG SNP) without 3′ACGP-3′ hairpin 3 Probe (531L 5′-FTTGGCQGCTGGGGCCC C P-3′ SNP) with 3′hairpin 4 Probe (531L 5′-FTTGGCQGCTGGGGCCCGP-3′ SNP) without 3′ hairpin

Designations: F stands for Threo-FAM; P stands for Phosphate; Q standsfor BHQ-2 quencher; and bolded underlined letters are bases that arechanged or added to form hairpin.

Platforms: LightCycler® 480 System

Real time PCR amplifications were performed using a set of forward andreverse primers and TaqMan® probes with a hairpin structure at the 3′end. Wild-type or Mutant plasmid DNA was tested at 1e6, 1e2, 1e3 and 1e1cp/PCR. The PCR reaction volume was 50 uL, and the master mix componentsand thermoprofile conditions are listed below. The amplifications wereperformed on the LC480 platform and the PCR growth curves were analyzedusing the ATF data analysis software. Results are shown in FIGS. 1through 8.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A kit for detecting a single nucleotide polymorphism(SNP) in a target nucleic acid in a sample, comprising: at least oneprimer comprising a first nucleic acid sequence specific to produce anamplification product comprising a region containing the SNP if thetarget nucleic acid is present in the sample; and a SNP specifichydrolysis probe comprising a second nucleic acid sequence complementaryto the region containing the SNP of the amplification product, the SNPspecific hydrolysis probe comprising a donor fluorescent moiety and anacceptor moiety of the donor fluorescent moiety, a 5′ end and a 3′ end,and a hairpin structure located on the 3′ end, the hairpin structurecomprising a region which is located on the 3′ end of the hairpinstructure that comprises one or more non-naturally occurring nucleotidesto produce the hairpin structure, wherein the acceptor moiety is in aninternal position of the SNP specific hydrolysis probe and is locatedclose to the 5′ end of the hairpin structure, the donor fluorescentmoiety is at the 5′ terminus, and the acceptor moiety is located withinno more than 5 nucleotides from the donor fluorescent moiety on thehydrolysis probe.
 2. The kit of claim 1, wherein the acceptor moiety isa quencher.
 3. The kit of claim 1, further comprising a polymeraseenzyme having 5′ to 3′ exonuclease activity.
 4. The kit of claim 1,wherein the first nucleic acid sequence of the primer and/or the secondnucleic acid sequence of the hydrolysis probe comprises/comprise atleast one modified nucleotide.
 5. The kit of claim 1, wherein the firstnucleic acid sequence of the primer and/or the second nucleic acidsequence of the hydrolysis probe has/have 40 or less than 40nucleotides.
 6. A single nucleotide polymorphism (SNP) specifichydrolysis probe comprising a nucleic acid sequence complementary to aSNP containing region of an amplification product, the SNP specifichydrolysis probe comprising a donor fluorescent moiety and an acceptormoiety of the donor fluorescent moiety, a 5′ end and a 3′ end, and ahairpin structure located on the 3′ end, the hairpin structurecomprising a region which is located on the 3′ end of the hairpinstructure that comprises one or more non-naturally occurring nucleotidesto produce the hairpin structure, wherein the acceptor moiety is in aninternal position of the SNP specific hydrolysis probe and is locatedclose to the 5′ end of the hairpin structure, the donor fluorescentmoiety is at the 5′ terminus, and the acceptor moiety is located withinno more than 5 nucleotides from the donor fluorescent moiety on thehydrolysis probe.
 7. The SNP specific hydrolysis probe of claim 6,wherein the acceptor moiety is a quencher.
 8. The SNP specifichydrolysis probe of claim 6, wherein the nucleic acid sequence of thehydrolysis probe comprises at least one modified nucleotide.
 9. The SNPspecific hydrolysis probe of claim 6, wherein the first nucleic acidsequence of the primer and/or the second nucleic acid sequence of thehydrolysis probe has/have 40 or less than 40 nucleotides.